Comparison of the deduced amino acid sequences of LadA and XdhA to other L-arabitol, xylitol and D-sorbitol dehydrogenases, as well as some putative dehydrogenases with unknown function demonstrated that these enzymes form distinct groups in the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240). Previously it was suggested that L-arabitol dehydrogenase might be the fungal orthologue of D-sorbitol dehydrogenase of higher eukaryotes [7]. However, the data in our study indicates that LAD, XDH and SDH are three distinct families, possibly originating from a common ancestor. Based on sequence identity (data not shown) and enzyme activity XDH appears to be more similar to SDH than LAD, as XDH but not LAD was shown to have significant activity on D-sorbitol [5], while SDH is significantly more active on xylitol than on L-arabitol (our study).
Interestingly, our study suggests that there is no clear fungal orthologue of SDH, based on BLAST and KEGG analysis. As the expression of A. niger ladA and xdhA appears highly specific for L-arabinose and D-xylose [5], it is unlikely that these enzymes are also acting as a sorbitol dehydrogenase for this fungus. A possible candidate sorbitol dehydrogenase might be the enzyme encoded by the uncharacterised gene from A. niger (An09g03900) that is in the groups that splits of the XDH branch in the tree. As orthologues for this gene were found in all tested fungi, it appears to encode a conserved function. However, bootstrap support for similarity of these enzymes to SDH is weak, indicating that no reliable prediction of function is possible based on these results.
The two homologues of LadA described for A. nidulans [7] cluster in the tree with LadA, but appear as separate branches. LadB appears to only be present in the Aspergilli, while LadC is present in most of the tested fungi. A previous study suggested the presence of a single L-arabitol dehydrogenase encoding gene involved in the L-arabinose catabolism [6], as a UV mutant of this gene was devoid of L-arabitol dehydrogenase activity. It is therefore likely that LadB and LadC have different biological functions f LadA.
Modelling of the structure from A. niger LadA and XdhA on human D-sorbitol dehydrogenase revealed a large number of amino acids that are conserved in all three types of dehydrogenases, including the residues involved in Zinc binding (H80, E81 and E166, numbers from LadA sequence) [13]. None of the residues that were conserved in L-arabitol and D-sorbitol dehydrogenases, but different in xylitol dehydrogenases were in close proximity of the substrate cleft. However, two of the residues (F62 and F302 from XdhA) that were conserved in xylitol and D-sorbitol dehydrogenases, but different in L-arabitol dehydrogenases (corresponding to M70 and Y318 from LadA) were located very close to the substrate, suggesting that they may be important for substrate specificity. As both XdhA and D-sorbitol dehydrogenase are active on D-sorbitol, whereas LadA has very little activity on this substrate [5] this could indicate that these residues are important for activity on D-sorbitol.
The M70F mutation of LadA of A. niger resulted in almost complete inactivation of the enzyme on a variety of substrates. The reason for this is not clear at this point, but a possible explanation could be that M70 in this particular enzyme influences the 3-dimensional structure; thus promoting enzyme activity. As the aim of this study was to identify residues important in substrate specificity, we did not further investigate this mutation.
The Y318F mutation of LadA resulted in increased affinity of the enzyme for D-sorbitol, while the Vmax and Kcat increased for L-arabitol and xylitol. Projection of the catalytic site of LAD, SDH and XDH predicts that the tyrosine residue in LAD and the phenylalanine in SDH and XDH are in exactly the same position (Fig. 3). This suggests that the OH group on the Y318 is the only structural difference between LadA and the Y318F mutant protein. This demonstrates that the presence of a phenylalanine at this position contributes significantly to D-sorbitol dehydrogenase activity. This OH-group probably affects positioning of D-sorbitol by hydrogen-bond formation in the substrate binding site, which prevents efficient catalysis in native A. niger LadA. The tyrosine residue does not affect affinity of LadA for L-arabitol and xylitol. However, the increased activity in the mutant suggests that the presence of the OH-group delays release of the products (L-xylulose and D-xylulose). D-sorbitol and xylitol differ structurally from L-arabitol with respect to positioning of the OH-group on C2 and C4, while D-sorbitol has an additional OH group at C5 compared to xylitol (Fig. 4). The increased affinity for D-sorbitol but not for L-arabitol or xylitol of the Y318F mutant may suggest that the presence of the OH group on Y318 in LadA interferes with the OH group on C5 of L-arabitol, resulting in a conformation for D-sorbitol in the active site that inhibits enzymatic conversion.
Genomes are continuously subjected to sequence mutations, resulting in evolution of species and biodiversity. Mutations that result in beneficial changes are likely to be maintained, while disadvantageous mutations will lose out in natural selection and therefore disappear again. The higher activity on L-arabitol of the Y318F mutant protein suggests an evolutionary advantage for this mutation with respect to conversion of this compound and therefore the efficiency of this metabolic pathway. This could indicate that this step in the pathway is not rate-limiting and therefore increased activity does not result in a biological advantage. Alternatively, since the increased activity is accompanied by a reduction in specificity this could provide selection against this mutation. It may be disadvantageous to convert other substrates simultaneously with L-arabitol, either due to competition for the enzyme or because the resulting product have a negative effect on growth.